Regulation of the Melanocyte Lineage by the AP2 Transcription Factor Family

干细胞维持方面的缺陷可能导致组织再生能力的丧失，或者相反， 在生长失调和癌症方面。干细胞如何在保留其再生能力的同时抵抗丧失其再生能力 对扩散的控制仍然知之甚少。黑素细胞干细胞(MSC)是一种理想的 对成体干细胞的一般研究。这种类型的细胞的凋亡或分化都会导致毛发 MITF编码黑素细胞发育的主要调节因子，它的灰白和突变一直是 被证明会导致小鼠头发变白。转录因子激活-增强子结合基因TFAP2A的突变 蛋白质2α)会导致鳃-眼-面综合征(BofS)，其中包括头发过早变白。 然而，TFAP2蛋白参与MSCs相关调控网络的机制 目前仍不清楚。基于斑马鱼、小鼠和培养的人类黑素细胞的大量初步数据， 我们的总体假设是AP-2活性调节黑素细胞的多能性、生长和分化。 和MSCs，与MITF平行工作，受KCTD15介导的SUMO作用调节。在目标1中，我们将 测试由两个平行对数冗余提供的细胞自主TFAP2活动是 对于MSCs的维持是必要的，并且BofS相关的TFAP2A变异体抑制这两个并列基因， 导致骨髓间充质干细胞异位分化和头发灰白。我们将在老鼠身上测试这一假设，方法是删除 相关的Tfap2与MSCs或MSC巢细胞中的Tfap2类似，并检测头发白化和MSC状态。 在目标2中，我们将检验这样的假设，即TFAP2同源基因作为先驱因子存在于基因座子集中 它与MITF结合，促进MITF介导的促进生长和分化的基因的激活。这 将需要从黑色素瘤细胞系中删除TFAP2同源基因，然后使用ATAC- SEQ，以及使用CHIP-SEQ结合MITF。我们将在原代黑素母细胞中进行平行实验 从斑马鱼胚胎中分离出来。在目标3中，我们将测试TFAP2A中的负反馈环的假设 信号转导对于维持和招募MSCs的正确平衡是必要的。我们发现一个 TFAP2-paralog反式激活活性的有效抑制剂KCTD15在黑素细胞中表达，并且 斑马鱼kctd15a/b突变体显示出多余的异位黑素细胞。我们将测试Kctd15- 体外实验表明，TFAP2A介导的总和甲基化可阻断其反式激活活性。我们将测试 Kctd15a通过取消其在黑素细胞中的表达而在控制黑素细胞分化中的作用 血统。我们还将测试TFAP2抑制在KCTD15的影响中的预测。 黑素细胞谱系。拟议研究的预期结果是对通路有更深入的了解 调节骨髓间充质干细胞生长和分化的平衡。对机制的理解，从而 MITF和TFAP2蛋白有助于MSC的维持，以及这些因素是如何调节的，将具有 广泛的影响，因为它们参与衰老机制和黑色素瘤的发生和发展。

Defects in the maintenance of stem cells can result in loss of the ability of tissues to regenerate or, conversely, in dysregulated growth and cancer. How the stem cell resists losing its regenerative qualities while retaining control of proliferation remains poorly understood. The melanocyte stem cell (MSC) is an ideal model for the study of adult stem cells in general. Either the apoptosis or differentiation of this cell type can cause hair graying, and mutations in MITF, which encodes the master regulator of melanocyte development, have been shown to cause hair graying in mice. Mutations in TFAP2A (Transcription Factor Activating-enhancer binding Protein 2 alpha) cause branchio-oculo-facial syndrome (BOFS), which includes premature hair graying. However, the mechanisms whereby TFAP2 proteins contribute to the relevant regulatory networks in MSCs remain unclear. Based on extensive preliminary data from zebrafish, mice, and cultured human melanocytes, our overall hypothesis is that AP-2 activity regulates pluripotency, growth and differentiation in melanocytes and MSCs, working in parallel with MITF and regulated by KCTD15-mediated sumoylation. In Aim 1 we will test the hypothesis that that cell-autonomous TFAP2 activity, provided redundantly by two paralogs, is necessary for the maintenance of MSCs, and that BOFS-associated TFAP2A variants inhibit both paralogs, leading to ectopic differentiation of MSCs and to hair graying. We will test this hypothesis in mice by deleting the relevant Tfap2 paralogs in MSCs or in cells of the MSC niche, and examining hair graying and MSC status. In Aim 2 we will test the hypothesis that TFAP2 paralogs serve as pioneer factors at a subset of the loci to which MITF binds, facilitating MITF-mediated activation of genes that promote growth and differentiation. This will entail deleting TFAP2 paralogs from a melanoma cell line and then assessing open chromatin using ATAC- SEQ, and binding of MITF using ChIP-SEQ. We will carry out parallel experiments in primary melanoblasts isolated from zebrafish embryos. In Aim 3 we will test the hypothesis that a negative feedback loop in TFAP2A signaling is necessary for the correct balance of maintenance and recruitment of MSCs. We found that a potent inhibitor of TFAP2-paralog transactivation activity, KCTD15, is expressed in melanocytes, and that zebrafish kctd15a/b mutants exhibit supernumerary, ectopic melanocytes. We will test the model that Kctd15- mediated sumoylation of Tfap2a blocks its transactivation activity, testing sumoylation in vitro. We will test kctd15a for a role in controlling melanocyte differentiation by abrogating its expression in the melanocyte lineage. We will also test the prediction that inhibition of TFAP2 mediates the effects of KCTD15 in the melanocyte lineage. The expected outcome of the proposed research is a deeper understanding of pathways that regulate the balance of growth and differentiation of MSCs. An understanding of the mechanisms whereby MITF and TFAP2 proteins contribute to MSC maintenance, and of how these factors are regulated, will have a broad impact as they participate in mechanisms of aging and in the initiation and progression of melanoma.